Tlr4-tlr7 ligand formulations as vaccine adjuvants

Abstract

A method to enhance an immune response in a mammal, and a composition comprising liposomes, a TLR4 agonist and a TLR7 agonist, are provided.

What is claimed is: 
     
         1 . A method to enhance an immune response in a mammal, comprising administering to a mammal in need thereof a composition comprising liposomes comprising an effective amount of a TLR4 agonist and a TLR7 agonist. 
     
     
         2 . The method of  claim 1  wherein the TLR4 agonist and a TLR7 agonist are administered simultaneously. 
     
     
         3 . The method of  claim 1  or  2  wherein the TLR4 agonist has formula (II): 
       
         
           
           
               
               
           
         
         wherein zI is an integer from 0 to 4, wherein z2 is an integer from 0 to 5, wherein R 5  is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R 6  is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R 7  is hydrogen, or substituted or unsubstituted alkyl, and wherein each R 8  is independently halogen, —CN, —SH, —OH, —COOH, —NH 2 , —CONH 2 , nitro, —CF 3 , —CCl 3 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. 
       
     
     
         4 . The method of any one of  claims 1  to  3  wherein the TLR7 agonist has formula (I): 
       
         
           
           
               
               
           
         
       
       wherein X 1  is —O—, —S—, or —NR c —;
 R 1  is hydrogen, (C 1 -C 10 )alkyl, substituted (C 1 -C 10 )alkyl, C 5-10 aryl, or substituted C 6-10 aryl, C 5-9 heterocyclic, substituted C 5-9 heterocyclic; 
 R c  is hydrogen, C 1-10 alkyl, or substituted C 1-10 alkyl; or R c  and R 1  taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; 
 each R 2  is independently —OH, (C 1 -C 6 )alkyl, substituted (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, substituted (C 1 -C 6 )alkoxy, —C(O)—(C 1 -C 6 )alkyl (alkanoyl), substituted —C(O)—(C 1 -C 6 )alkyl, —C(O)—(C 6 -C 10 )aryl (aroyl), substituted —C(O)—(C 6 -C 10 )aryl, —C(O)OH (carboxyl), —C(O)O(C 1 -C 6 )alkyl (alkoxycarbonyl), 
 
       substituted —C(O)O(C 1 -C 6 )alkyl, —NR a R b , —C(O)NR a R b  (carbamoyl), halo, nitro, or cyano, or R 2  is absent:
 each R a  and R b  is independently hydrogen, (C 1 -C 6 )alkyl, substituted (C 1 -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, substituted (C 3 -C 8 )cycloalkyl, (C 1 -C 6 )alkoxy, substituted (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, substituted (C 1 -C 6 )alkanoyl, aryl, aryl(C 1 -C 6 )alkyl, Het, Het (C 1 -C 6 )alkyl, or (C 1 -C 6 )alkoxycarbonyl; 
 wherein the substituents on any alkyl, aryl or heterocyclic groups are hydroxy, C 1-6 alkyl, hydroxyC 1-6 alkylene, C 1-6 alkoxy, C 3-6 cycloalkyl, C 1-6 alkoxyC 1-6 alkylene, amino, cyano, halo, or aryl; 
 n is 0, 1, 2, 3 or 4; 
 X 2  is a bond or a linking group; and 
 in one embodiment, R x  is a phospholipid comprising one or two carboxylic esters; 
 or a tautomer thereof; 
 or a pharmaceutically acceptable salt or solvate thereof 
 
     
     
         5 . The method of any one of  claims 1  to  4  wherein the liposomes comprise PC, DOPC, or DSPC. 
     
     
         6 . The method of any one of  claims 1  to  4  wherein the liposomes comprise cholesterol. 
     
     
         7 . The method of any one of  claims 1  to  6  further comprising administering one or more immunogens. 
     
     
         8 . The method of  claim 7  wherein the immunogen is a microbial immunogen. 
     
     
         9 . The method of  claim 8  wherein the microbe is a virus or a bacteria. 
     
     
         10 . The method of any one of  claims 7  to  9  wherein the liposomes comprise the one or more immunogens. 
     
     
         11 . The method of any one of  claims 1  to  10  wherein the mammal is a human. 
     
     
         12 . The method of any one of  claims 1  to  11  wherein the amount of the TLR7 agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol. 
     
     
         13 . The method of any one of  claims 1  to  12  wherein the amount of the TLR4 agonist is about 2 to 20 umol, about 20 nmol to 2 umol, or about 2 umol to about 100 umol. 
     
     
         14 . The method of any one of  claims 1  to  13  wherein the ratio of TLR7 to TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200. 
     
     
         15 . The method of any one of  claims 1  to  13  wherein the composition is injected, intramuscularly administered, intranasally administered or intravenously administered. 
     
     
         16 . The method of any one of  claims 1  to  15  wherein the liposomes comprise DOPC and cholesterol. 
     
     
         17 . A pharmaceutical formulation comprising liposomes, a TLR4 agonist and a TLR7 agonist. 
     
     
         18 . The formulation of  claim 17  wherein the liposome comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG-2000 PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-glycero-3-phosphocholine (18:1-12:0 NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphocholine (16:0-12:0 NBD PC), and mixtures thereof; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, or a mixture thereof. 
     
     
         19 . The formulation of  claim 17  wherein the liposome comprises DOPC, cholesterol or combinations thereof. 
     
     
         20 . The formulation of any one of  claims 17  to  19  wherein the amount of the TLR7 agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol. 
     
     
         21 . The formulation of any one of  claims 17  to  20  wherein the amount of the TLR4 agonist is about 2 nmol to 20 umol, about 20 nmol to 2 umol, or about 2 umol to about 100 umol. 
     
     
         22 . The formulation of any one of  claims 17  to  21  wherein the ratio of TLR7 to TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200. 
     
     
         23 . The formulation of any one of  claims 17  to  22  wherein the TLR7 agonist comprises a compound of Formula (I): 
       
         
           
           
               
               
           
         
       
       wherein X 1  is —O—, —S—, or —NR c —;
 R 1  is hydrogen, (C 1 -C 10 )alkyl, substituted (C 1 -C 10 )alkyl, C 5-10 aryl, or substituted C 6-10 aryl, C 5-9 heterocyclic, substituted C 5-9 heterocyclic; 
 R c  is hydrogen, C 1-10 alkyl, or substituted C 1-10 alkyl: or R c  and R 1  taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; 
 each R 2  is independently —OH, (C 1 -C 6 )alkyl, substituted (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, substituted (C 1 -C 6 )alkoxy, —C(O)—(C 1 -C 6 )alkyl (alkanoyl), substituted —C(O)—(C 1 -C 6 )alkyl, —C(O)—(C 6 -C 10 )aryl (aroyl), substituted —C(O)—(C 6 -C 10 )aryl, —C(O)OH (carboxyl), —C(O)O(C 1 -C 6 )alkyl (alkoxycarbonyl), 
 
       substituted —C(O)O(C 1 -C 6 )alkyl, —NR a R b , —C(O)NR a R b (carbamoyl), halo, nitro, or cyano, or R 2  is absent;
 each R a  and R b  is independently hydrogen, (C 1 -C 6 )alkyl, substituted (C 1 -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, substituted (C 3 -C 8 )cycloalkyl, (C 1 -C 6 )alkoxy, substituted (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, substituted (C 1 -C 6 )alkanoyl, aryl, aryl(C 1 -C 6 )alkyl, Het, Het (C 1 -C 6 )alkyl, or (C 1 -C 6 )alkoxycarbonyl; 
 wherein the substituents on any alkyl, aryl or heterocyclic groups are hydroxy, C 1-6  alkyl, hydroxyC 1-6 alkylene, C 1-6 alkoxy, C 3-6 cycloalkyl, C 1-6 alkoxyC 1-6 alkylene, amino, cyano, halo, or aryl; 
 n is 0, 1, 2, 3 or 4; 
 X2 is a bond or a linking group; and 
 R 3  is a phospholipid comprising one or two carboxylic esters; 
 
       or a tautomer thereof; 
       or a pharmaceutically acceptable salt or solvate thereof. 
     
     
         24 . The formulation of  claim 23  wherein R 3  in formula (I) comprises 
       
         
           
           
               
               
           
         
       
       wherein R 11  and R 12  are each independently a hydrogen or an acyl group, R 13  is a negative charge or a hydrogen, and m is 1 to 8, wherein a wavy line indicates a position of bonding, wherein an absolute configuration at the carbon atom bearing OR 12  is R, S, or any mixture thereof. 
     
     
         25 . The formulation of  claim 23  or  24  wherein m is 1 or wherein R 11  and R 12  are each oleoyl groups. 
     
     
         26 . The formulation of any one of  claims 23  to  25  wherein the phospholipid of R 3  comprises two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof. 
     
     
         27 . The formulation of any one of  claims 23  to  26  wherein the phospholipid of R 3  comprises two carboxylic esters and the carboxylic esters of are the same or different. 
     
     
         28 . The formulation of  claim 27  wherein each carboxylic ester of the phospholipid is a C17 carboxylic ester with a site of unsaturation at C8-C9. 
     
     
         29 . The formulation of  claim 27  wherein each carboxylic ester of the phospholipid is a C18 carboxylic ester with a site of unsaturation at C9-C10. 
     
     
         30 . The formulation of any one of  claims 23  to  29  wherein X 2  is a bond or a chain having one to about 10 atoms in a chain wherein the atoms of the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, wherein any carbon atom can be substituted with oxo, and wherein any sulfur atom can be substituted with one or two oxo groups. 
     
     
         31 . The formulation of any one of  claims 23  to  30  wherein R 3  comprises dioleoylphosphatidyl ethanolamine (DOPE). 
     
     
         32 . The formulation of any one of  claims 23  to  31  wherein R 3  is 1,2-dioleoyl-sn-glycero-3-phospho ethanolamine and X 2  is C(O). 
     
     
         33 . The formulation of any one of  claims 23  to  32  wherein X 1  is oxygen. 
     
     
         34 . The formulation of any one of  claims 23  to  33  wherein X 1  is O, R 1  is C 1-4 alkoxy-ethyl, n is O, X 2  is carbonyl, and R 3  is 1,2-dioleoylphosphatidyl ethanolamine (DOPE). 
     
     
         35 . The formulation of any one of  claims 23  to  33  wherein the compound of Formula (I) is: 
       
         
           
           
               
               
           
         
       
     
     
         36 . The formulation of any one of  claims 23  to  33  wherein the compound of Formula (I) is 
       
         
           
           
               
               
           
         
       
     
     
         37 . The formulation of any one of  claims 17  to  36  wherein the TLR4 agonist comprises formula (II): 
       
         
           
           
               
               
           
         
         wherein zI is an integer from 0 to 4, wherein z2 is an integer from 0 to 5, wherein R 5  is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R 6  is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R 7  is hydrogen, or substituted or unsubstituted alkyl, and wherein each R 8  is independently halogen, —CN, —SH, —OH, —COOH, —NH 2 , —CONH 2 , nitro, —CF 3 , —CCl 3 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. 
       
     
     
         38 . The formulation of  claim 37  wherein z2 is 1, 2 or 3. 
     
     
         39 . The formulation of  claim 37  or  38  wherein z1 is 1 or 2. 
     
     
         40 . The formulation of  claim 37  or  38  wherein z1 is 0. 
     
     
         41 . The formulation of any one of  claims 37  to  40  wherein R 5  is substituted or unsubstituted aryl. 
     
     
         42 . The formulation of any one of  claims 37  to  41  wherein R 6  is substituted or unsubstituted cycloalkyl. 
     
     
         43 . The formulation of any one of  claims 37  to  42  wherein R 7  is substituted or unsubstituted alkyl. 
     
     
         44 . The formulation of any one of  claims 37  to  39  or  40  to  43  wherein z1=1 and R 8  is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl.